Microglial and astroglial activation by 3,4-methylenedioxymethamphetamine (MDMA) in mice depends on S(+) enantiomer and is associated with an increase in body temperature and motility

Authors

  • Lucia Frau,

    1. Department of Biomedical Sciences, Section of Neuropsychopharmacology, University of Cagliari, Cagliari, Italy
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  • Nicola Simola,

    1. Department of Biomedical Sciences, Section of Neuropsychopharmacology, University of Cagliari, Cagliari, Italy
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  • Antonio Plumitallo,

    1. Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy
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  • Micaela Morelli

    Corresponding author
    1. Centre of Excellence for Neurobiology of Dependence, University of Cagliari, Cagliari, Italy
    2. CNR, Institute of Neuroscience, Cagliari, Italy
    • Department of Biomedical Sciences, Section of Neuropsychopharmacology, University of Cagliari, Cagliari, Italy
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Address correspondence and reprint requests to Prof. Micaela Morelli, Department of Biomedical Sciences, University of Cagliari, Via Ospedale 72, 09124 Cagliari, Italy. E-mail: morelli@unica.it

Abstract

Evidence is accumulating to suggest that 3,4-methylenedioxymethamphetamine (MDMA) has neurotoxic and neuroinflammatory properties. MDMA is composed of two enantiomers with different biological activities. In this study, we evaluated the in vivo effects of S(+)-MDMA, R(−)-MDMA, and S(+)-MDMA in combination with R(−)-MDMA on microglial and astroglial activation compared with racemic MDMA, by assessment of complement type 3 receptor (CD11b) and glial fibrillary acidic protein (GFAP) immunoreactivity in the mouse striatum, nucleus accumbens, motor cortex, and substantia nigra. Motor activity and body temperature were also measured, to elucidate the physiological modifications paired with the observed glial changes. Similar to racemic MDMA (4 × 20 mg/kg), S(+)-MDMA (4 × 10 mg/kg) increased both CD11b and GFAP in the striatum, although to a lower degree, whereas R(−)-MDMA (4 × 10 mg/kg) did not induce any significant glial activation. Combined administration of S(+) plus R(−)-MDMA did not induce any further activation compared with S(+)-MDMA. In all other areas, only racemic MDMA was able to slightly activate the microglia, but not the astroglia, whereas enantiomers had no effect, either alone or in combination. Racemic MDMA and S(+)-MDMA similarly increased motor activity and raised body temperature, whereas R(−)-MDMA affected neither body temperature nor motor activity. Interestingly, the increase in body temperature was correlated with glial activation. The results show that no synergism, but only additivity of effects, is caused by the combined administration of S(+)- and R(−)-MDMA, and underline the importance of investigating the biochemical and behavioral properties of the two MDMA enantiomers to understand their relative contribution to the neuroinflammatory and neurotoxic effects of MDMA.

Abbreviations used
AccCore

nucleus accumbens core

AccSh

nucleus accumbens shell

CD11b

complement type 3 receptor

GFAP

glial fibrillary acidic protein

MDMA

3,4-methylenedioxymethamphetamine

SNc

substantia nigra pars compacta

SNr

substantia nigra pars reticulata

3,4-methylenedioxymethamphetamine (MDMA), otherwise called ‘ecstasy’, is an amphetamine derivative with psychostimulant and rewarding properties (Schenk 2009), that can also induce neurotoxic effects (Rattray 1991; Johnson et al. 2002; Green et al. 2003; Hayat et al. 2006; Granado et al. 2008a, b; Khairnar et al. 2010). MDMA is composed of two enantiomers, raising the concern about the relative potential neurotoxic effects of the S(+)- and R(−)-MDMA forms.

Several studies have demonstrated that the two enantiomers have different biological activities (Fantegrossi et al. 2003, 2009; Acquas et al. 2007; Fantegrossi 2008; Young and Glennon 2008). For this reason, in vivo studies with the MDMA racemate may not be informative with regard to the potential neurotoxic effects of the two enantiomers. The evaluation of the in vivo activity of S(+)- and R(−)-MDMA is therefore particularly valuable, in view of the large use and varied composition of ecstasy pills sold on the illegal market (see Schifano 2004; for a review).

The in vivo pharmacology of MDMA and its enantiomers may differ depending on the species under investigation, with S(+)-MDMA usually being more potent than R(−)-MDMA.

In mice, S(+)-MDMA was found to induce motor activation more efficiently than R(−)-MDMA (Young and Glennon 2008), whereas in rats, S(+)-MDMA and (+/−)-MDMA were found to be more potent than R(−)-MDMA in contrasting akinesia (Lebsanft et al. 2005), increasing dopamine release, and activating phosphorylated extracellular signal regulated kinase, a marker correlated with post-synaptic dopaminergic activity (Acquas et al. 2007). Moreover, evidence has been obtained to suggest that S(+)-MDMA, but not R(−)-MDMA, increases body temperature (Fantegrossi et al. 2003). In contrast, in rhesus monkeys, few differences were observed in the capacities of racemic MDMA and its enantiomers to maintain behaviors such as self-administration, showing higher tolerance to the reinforcing effects of MDMA racemate or the R(−)-enantiomer, whereas S(+)-MDMA was less susceptible to this effect (Fantegrossi 2008). In addition, maximum and average body temperatures were found to be elevated by (+/−)-MDMA and by each enantiomer. This hyperthermia occurred despite a lack of increased locomotor activity, showing no dependency on motor activation (Taffe et al. 2006). Interestingly, in rhesus monkeys, S(+)-MDMA interacts with the dopamine transporter, whereas negligible dopamine transporter occupancy was observed following the administration of R(−)-MDMA (Fantegrossi 2008). Taken together, these findings suggest that the ability of S(+)- and R(−)-MDMA enantiomers to induce biological activity may significantly vary, depending on the species under investigation and on the type of activity evaluated.

The majority of published studies on MDMA chirality have focused on the behavioral differences and the relative activity on the monoamine system produced by the two different enantiomeric forms. However, besides producing behavioral changes correlated with the monoaminergic system, MDMA is known to induce neurotoxic effects correlated with inflammatory processes, such as glial activation and an increase in body temperature (Miller and O'Callaghan 1995; Zhang et al. 2006; Granado et al. 2008b; Touriño et al. 2010). Interestingly, recent evidence, together with several previous studies, has demonstrated that glial activation actively participates in the events inducing neuronal damage, instead of being just a reactive process (Halliday and Stevens 2011).

To study the relative inflammatory potential of S(+)- and R(−)-MDMA enantiomers, this study was designed to assess microglial and astroglial activation in different regions of the mouse brain by studying complement type 3 receptor (CD11b) and glial fibrillary acidic protein (GFAP) immunoreactivity. Since, as mentioned above, MDMA increases body temperature and stimulates motor activity in rodents, we evaluated the modifications in these parameters in treated mice, as an index of pharmacological activity. Finally, since the increase in body temperature has been suggested to be a causative factor in MDMA-induced neuroinflammation (Touriño et al. 2010), we evaluated whether a direct correlation exists between body temperature modifications and glial activation.

As described in previous studies (Granado et al. 2008b; Khairnar et al. 2010), a protocol of acute repeated administration of MDMA was utilized to obtain a consistent enhanced activation of the glial cells (Granado et al. 2008b; Khairnar et al. 2010). Moreover, the doses of (+/−)-MDMA used were similar to those of previous studies reporting glial activation (Khairnar et al. 2010), while, based on the lack of optical activity of the (+/−) MDMA, indicative of a 1:1 ratio between MDMA enantiomers, S(+)- and R(−)-MDMA doses were calculated as half of the racemic MDMA dose.

Materials and methods

Animals

Adult male 12-week-old C57BL/6J mice (Charles River, Calco, Italy) were used in the study. Mice were housed in groups of five per cage (length, 42 cm; width, 24 cm; height, 15 cm) in a temperature- (21 ± 1°C), humidity- (55 ± 10%), and light-cycle-controlled room. Lights were ON between 08:00 and 20:00 h, and the experiments took place during the light phase. Food and water were available ad libitum, except during the experiments.

All experimental procedures were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and approved by the Ethical Committee of the University of Cagliari. Efforts were made to keep the number of animals used to the lowest possible, and to minimize animal suffering.

Drugs

(+/−)-MDMA-HCl, S(+)-MDMA-HCl, and R(−)-MDMA-HCl (synthesized by Prof. Antonio Plumitallo, Department of Life and Environmental Sciences, University of Cagliari) were dissolved in saline and administered intraperitoneally (i.p.) in a volume of 10 ml/kg. Racemic MDMA was synthesized from 1-(3,4-methylenedioxyphenyl)-propanone (Borch et al. 1971), which was subject to reductive amination with methylamine in the presence of sodium cyanoborohydride. MDMA enantiomers were synthesized starting from 1-(3,4-methylenedioxyphenyl)-propanone (Hass et al. 1950). Reductive amination was performed by using R- or S-α-methylbenzylamine and W2 Raney Ni. This reaction led to N-α-phenylethylamine intermediates (Nichols et al. 1986), which were then subject to methylation and N-debenzylation, by using Pd–C as a catalyst under a pressure of 3 atm (Fig. 1). (+/−)-MDMA (optically inactive racemate), S(+)-MDMA, and R(−)-MDMA were all obtained as hydrochlorides.

Figure 1.

Synthesis of 3,4-methylenedioxymethamphetamine (MDMA) and its enantiomers. (+/−)-MDMA (a) was synthesized by reductive amination of 1-(3,4-methylenedioxyphenyl)-propanone (b) with methylamine. S(+)-MDMA (c) and R(−)-MDMA (d) were synthesized from (b), which was subject to reductive amination by using S- or R-α-methylbenzylamine, respectively.

Treatments

On the basis of the lack of optical activity of the (+/−)-MDMA used in this study, indicative of a 1 : 1 ratio between MDMA enantiomers, we administered a dose of R(−) and S(+)-MDMA calculated as the half of the racemic MDMA dose.

Mice were treated with vehicle (saline), (+/−)-MDMA (4 × 20 mg/kg, 2 h intervals, i.p.), S(+)-MDMA (4 × 10 mg/kg, 2 h intervals, i.p.), R(−)-MDMA (4 × 10 mg/kg, 2 h intervals, i.p.), or the combination of S(+)-MDMA (4 × 10 mg/kg, 2 h intervals, i.p.) plus R(−)-MDMA (4 × 10 mg/kg, 2 h intervals, i.p.). Each animal received a total of four administrations. Pharmacological treatments were performed in a room kept at the constant temperature of 21 ± 1°C.

Forty-eight hours after the last vehicle or drug administration, mice were anesthetized with chloral hydrate (400 mg/kg, i.p.), transcardially perfused with paraformaldehyde (4% in 0.1 M phosphate buffer, pH 7.4) and used for immunohistochemistry studies. Rectal temperature was recorded 1 h after each vehicle or drug administration.

Immunohistochemistry

Sections (50 μm thick) from the nucleus accumbens, shell (AccSh) and core (AccCore), M1 and M2 areas of motor cortex, striatum, and substantia nigra, both pars compacta (SNc) and pars reticulata (SNr) were coronally cut on a vibratome and immunoreacted with CD11b and GFAP antibodies [monoclonal rat anti-mouse CD11b, 1 : 1000 (Serotec, Oxford, UK) and monoclonal mouse anti-GFAP, 1 : 400 (Sigma, Milan, Italy)] and proper secondary antibodies [goat anti-rat IgG for CD11b and goat anti-mouse IgG for GFAP (both from Vector, Peterborough, UK)]. For visualization, the avidin-peroxidase protocol (ABC; Vector) was applied, using 3,3′-diaminobenzidine (Sigma) as chromogen, according to Frau et al. (2011). Sections were mounted on gelatine-coated slides, dehydrated and coverslipped.

Analysis of GFAP immunoreactivity

Images were digitized (videocamera PixeLink PL-A686, PixeLink Microscopy Cameras, Ottawa, Ontario, Canada) under constant light conditions. Sections were captured at 20× magnification. For each animal, three sections from the nucleus accumbens (A = 1.70 mm; A = 1.42 mm; A = 1.18 mm), motor cortex (A = 1.70 mm; A = 1.42 mm; A = 0.26 mm), substantia nigra (A = −2.92 mm; A = −3.28 mm; A = −3.64 mm), and striatum (A = 1.10 mm; A = 0.74 mm; A = 0.38 mm) were analyzed. Sections were taken from bregma, according to the Mouse Brain Atlas, Paxinos and Franklin 2001). The analysis was performed for each section, and included: (i) two portions of AccSh and AccCore, left and right; (ii) one portion from the M1 and one portion from the M2 motor cortex, left and right; (iii) the whole left and right SNc; (iv) the whole left and right SNr; (v) one portion from the dorsolateral striatum and one portion from the ventromedial striatum, left and right. As no differences were observed between the M1 and M2 motor cortex areas and between the dorsolateral and ventromedial striatum, the values were pooled together for each area. Images were captured using the PixeLink capture camera. The PixeLink software was used to carry out the analysis, which was performed by counting the total number of GFAP-positive cells in the selected areas. The obtained value was first normalized with respect to vehicle, and values from different section levels were averaged thereafter.

Analysis of CD11b immunoreactivity

Images were digitized in a gray scale using the PixeLink capture camera, and CD11b immunostaining was evaluated with the analysis program Scion Image. In each section, the selected areas (i) two portions of the AccSh and AccCore; (ii) one portion from the M1 and one portion from the M2 motor cortex; (iii) one portion from the dorsal SNc and one portion from the ventral SNc; (iv) one portion from the dorsal SNr and one portion from the ventral SNr; (v) one portion from the dorsolateral striatum and one portion from the ventromedial striatum, left and right, were analyzed with the same procedure used for GFAP analysis. As, similar to the GFAP analysis, no differences were observed between the M1 and M2 motor cortex, the dorsal and ventral SNc, the dorsal and ventral SNr, and the dorsolateral and ventromedial striatum, the values from these portions were pooled together for each area. A threshold, the value of which was set above the mean value ± SEM of the background, was applied for background correction. Inside each frame, the area occupied by gray values above the threshold was automatically calculated. For each level, the obtained value was first normalized with respect to vehicle, and values from the different levels were averaged thereafter.

Temperature recording

Body temperature was measured using a rectal probe (BRET-3) digital thermometer [MicroTherma 2T Hand Held Thermometer (2Biological Instruments, Besozzo, Varese, Italy)]. Baseline temperature was recorded prior to the first vehicle or drug administration, to ascertain that no significant differences existed among the experimental groups and then 1 h after each vehicle or drug administration. Temperature was recorded by holding each mouse at the base of the tail and inserting the probe past the rectum into the colon for 4–5 s, until rectal temperature was maintained for 3 s.

Motor activity measurement

Motor activity was evaluated in a group of mice different from that used for immunohistochemical evaluation. Measurement of motor activity was carried out in a quiet, isolated room. Each mouse was placed individually in a cage (length, 47 cm; width, 27 cm; height, 19 cm) equipped with two pairs of infrared photocell emitters and detectors located along the long axis (Opto-Varimex; Columbus Instruments, Columbus, OH, USA). Two kinds of motor activity were registered: horizontal locomotion along the long axis of the cage, which caused interruption of different beams, and total motor activity, including horizontal locomotion plus vertical activity, the latter causing interruption of the same infrared beam. Mice were habituated to the cages for 1 h before the first vehicle or drug administration. Motor activity was evaluated for eight consecutive hours, starting after the first injection. Activity counts were taken every 20 min, for a total of six evaluations (cumulative time: 2 h) within each drug administration (I, II, III, and IV). Pharmacological treatments and motor activity measurement were performed in a room kept at the constant temperature of 21 ± 1°C.

Statistics

Data from the immunohistochemical analysis were statistically compared with a one-way anova, followed by Tukey's post-hoc test. Data from temperature and motor activity measurements were analyzed with a two-way anova, followed by Newman–Keuls post-hoc test. Linear regression analysis was used to correlate the increase in body temperature (calculated as the average value over the four administrations) and activation of the microglia and astroglia in the striatum. Correlation analysis did not include the glial activation detected in the other brain areas studied (nucleus accumbens, substantia nigra, and motor cortex). Thus, the analysis was narrowed to the striatum because it was the only area where a robust activation of both astroglia and microglia could be observed after the administration of either MDMA enantiomer, as well as of (+/−)-MDMA. In addition, brain areas other than the striatum displayed glial activation only in response to (+/−)-MDMA but not to MDMA enantiomers, and only microglia, but not astroglia, was affected. All results were considered significant at p < 0.05.

Results

Microglial activation in the striatum

In vehicle-treated mice, resting microglia and very low levels of CD11b were observed. (+/−)-MDMA (4 × 20 mg/kg) induced a significant increase in CD11b immunoreactivity, compared with vehicle. S(+)-MDMA (4 × 10 mg/kg) elicited a significant increase in CD11b levels, compared with vehicle; nevertheless, this increase was lower than that observed after (+/−)-MDMA. R(−)-MDMA (4 × 10 mg/kg) did not induce any changes in CD11b immunoreactivity, compared with vehicle. When administered in combination, S(+)-MDMA plus R(−)-MDMA induced an increase in microglial reactivity which tended to be higher than the increase induced by S(+)-MDMA alone. In addition, the combination of S(+)-MDMA plus R(−)-MDMA was not significantly different from (+/−)-MDMA (Fig. 2).

Figure 2.

Effect of racemic 3,4-methylenedioxymethamphetamine (MDMA) and MDMA enantiomers on microglial activation in the striatum. Representative sections and histograms of striatum immunostained for CD11b. Mice were treated with vehicle (saline, i.p., n = 12), (+/−)-MDMA (4 × 20 mg/kg, i.p., n = 14), S(+)-MDMA (4 × 10 mg/kg, i.p., n = 16), R(−)-MDMA (4 × 10 mg/kg, i.p., n = 11), or S(+)-MDMA plus R(−)-MDMA (n = 5) and killed 48 h after the last vehicle or drug administration. The area occupied by gray values above a threshold was calculated and expressed as square pixels and as a percentage of staining in vehicle-treated mice. Values are expressed as mean ± SEM. *p < 0.05 compared with vehicle-treated mice; ^p < 0.05 compared with (+/−)-MDMA-treated mice, by Tukey's post-hoc test. Scale bar, 50 μm.

Microglial activation in the nucleus accumbens, motor cortex, and substantia nigra

Similar to that observed in the striatum, resting microglia and very low levels of CD11b were present in the AccSh and AccCore, M1 and M2 motor cortex, and SNc and SNr of vehicle-treated mice. (+/−)-MDMA (4 × 20 mg/kg) induced a significant increase in CD11b immunoreactivity in both the AccSh and AccCore (Fig. 3a and b), motor cortex (Fig. 3c), and SNc (Fig. 3d), compared with vehicle-treated mice, leaving the levels of CD11b in the SNr unaltered (data not shown). On the other hand, S(+)-MDMA (4 × 10mg/kg), R(−)-MDMA (4 × 10 mg/kg), and the combination of S(+)-MDMA plus R(−)-MDMA did not induce any increase in microglial reactivity in all the areas analyzed, compared with vehicle-treated mice (Fig. 3).

Figure 3.

Effect of racemic 3,4-methylenedioxymethamphetamine (MDMA) and MDMA enantiomers on microglial activation in the nucleus accumbens, motor cortex, and SNc. Histograms of AccSh (a), AccCore (b), motor cortex (c) and SNc (d) immunostained for CD11b. Mice were treated with vehicle (saline, i.p., n = 12), (+/−)-MDMA (4 × 20 mg/kg, i.p., n = 14), S(+)-MDMA (4 × 10 mg/kg, i.p., n = 16), R(−)-MDMA (4 × 10 mg/kg, i.p., n = 11), or S(+)-MDMA plus R(−)-MDMA (n = 5) and killed 48 h after the last vehicle or drug administration. The area occupied by gray values above a threshold was calculated and expressed as square pixels and as a percentage of staining in vehicle-treated mice. Values are expressed as mean ± SEM. *p < 0.05 compared with vehicle-treated mice; ^p < 0.05 compared with (+/−)-MDMA-treated mice, by Tukey's post-hoc test. Scale bar, 50 μm.

Astroglial activation in the striatum

Vehicle-treated mice displayed few GFAP-positive cells. (+/−)-MDMA (4 × 20 mg/kg) induced a significant increase in GFAP immunoreactivity, compared with vehicle-treated mice. S(+)-MDMA (4 × 10 mg/kg) induced an increase in the number of GFAP-positive cells, compared with vehicle, but this increase was lower than that caused by (+/−)-MDMA. R(−)-MDMA (4 × 10 mg/kg) did not induce any change in the number of GFAP-positive cells, compared with vehicle. The combination of enantiomers increased GFAP immunoreactivity in a fashion similar to S(+)-MDMA (Fig. 4).

Figure 4.

Effect of racemic 3,4-methylenedioxymethamphetamine (MDMA) and MDMA enantiomers on astroglial activation. Representative sections and histograms of striatum immunostained for glial fibrillary acidic protein (GFAP). Mice were treated with vehicle (saline, i.p., n = 12), (+/−)-MDMA (4 × 20 mg/kg, i.p., n = 14), S(+)-MDMA (4 × 10 mg/kg, i.p., n = 16), R(−)-MDMA (4 × 10 mg/kg, i.p., n = 11), or S(+)-MDMA plus R(−)-MDMA (n = 5) and killed 48 h after the last vehicle or drug administration. The graph shows the number of GFAP-positive cells expressed as a percentage of vehicle-treated mice. Values are expressed as mean ± SEM. *p < 0.05 compared with vehicle-treated mice; ^p < 0.05 compared with (+/−)-MDMA-treated mice, by Tukey's post-hoc test. Scale bar, 50 μm.

Astroglial activation in the nucleus accumbens, motor cortex, and substantia nigra

Similar to that observed in the striatum, few GFAP-positive cells could be detected in vehicle-treated mice. (+/−)-MDMA (4 × 20 mg/kg) did not induce any increase in GFAP immunoreactivity, compared with vehicle-treated mice in any of the areas under investigation. Similar results were obtained after S(+)-MDMA (4 × 10 mg/kg), R(−)-MDMA (4 × 10 mg/kg), and their combined administration (data not shown).

Body temperature

Rectal temperature recorded before the beginning of treatments revealed no differences in basal values among the experimental groups.

Treatment with vehicle did not induce any change in rectal temperature compared with basal values. (+/−)-MDMA (4 × 20 mg/kg) induced a significant increase in body temperature compared with vehicle-treated mice after each drug administration. S(+)-MDMA (4 × 10 mg/kg) also increased body temperature, although to a lower extent than (+/−)-MDMA on administrations II and III. R(−)-MDMA (4 × 10 mg/kg) did not affect body temperature. The combination of S(+)-MDMA and R(−)-MDMA increased body temperature similar to (+/−)-MDMA and S(+)-MDMA (Fig. 5). Moreover, the existence of a positive correlation between the increase in body temperature and the activation of microglia and astroglia in the striatum was observed (Fig. 6).

Figure 5.

Effect of racemic 3,4-methylenedioxymethamphetamine (MDMA) and MDMA enantiomers on body temperature. The graph reports the variations in body temperature induced by racemic (+/−)-MDMA and MDMA enantiomers. Mice treated with vehicle (saline, i.p., n = 12), (+/−)-MDMA (4 × 20 mg/kg, i.p., n = 14), S(+)-MDMA (4 × 10 mg/kg, i.p., n = 16), R(−)-MDMA (4 × 10 mg/kg, i.p., n = 11), or S(+)-MDMA plus R(−)-MDMA (n = 5). Values are expressed as mean ± SEM. Body temperature was found to be significantly elevated following (+/−)-MDMA, S(+)-MDMA and S(+)-MDMA plus R(−)-MDMA compared with vehicle and R(−)-MDMA (p < 0.05 compared with vehicle-treated mice; ^p < 0.05 compared with (+/−)-MDMA-treated mice, by Newmann–Keuls post-hoc test).

Figure 6.

Correlation between body temperature and glial activation. The Figure shows the existence of a positive correlation between the increase in body temperature (calculated as the average value over the four administrations) and the activation of microglia (a) and astroglia (b). Mice treated with vehicle (saline, i.p., n = 12), (+/−)-3,4-methylenedioxymethamphetamine (MDMA) (4 × 20 mg/kg, i.p., n = 14), S(+)-MDMA (4 × 10 mg/kg, i.p., n = 16), R(−)-MDMA (4 × 10 mg/kg, i.p., n = 11), or S(+)-MDMA plus R(−)-MDMA (n = 5). The body temperature values from the different experimental groups were pooled together for the correlation analysis.

Motor activity

(+/−)-MDMA (4 × 20 mg/kg) induced a significant increase in both the total motor (Fig. 7a) and locomotor activity (Fig. 7b), compared with vehicle. Similar effects were induced by S(+)-MDMA (4 × 10 mg/kg), whereas R(−)-MDMA (4 × 10 mg/kg) did not modify the total motor and locomotor activity, compared with vehicle. The combination of S(+)-MDMA plus R(−)-MDMA, similar to (+/−)-MDMA and S(+)-MDMA, induced an increase in both the total motor and locomotor activity (Fig. 7).

Figure 7.

Effect of racemic 3,4-methylenedioxymethamphetamine (MDMA) and MDMA enantiomers on motor activity. The graphs show the total motor (a) and locomotor activity (b) counts registered over the course of the treatment, reported as total counts calculated for each drug administration Activity counts were taken every 20 min for a total of six evaluations (cumulative time: 2 h) within each drug administration (I, II, III, and IV). Mice were treated with vehicle (saline, i.p., n = 5), (+/−)-MDMA (4 × 20 mg/kg, i.p., n = 5), S(+)-MDMA (4 × 10 mg/kg, i.p., n = 5), R(−)-MDMA (4 × 10 mg/kg, i.p., n = 5), or S(+)-MDMA plus R(−)-MDMA (n = 5). Values are expressed as mean ± SEM. *p < 0.05 compared with vehicle-treated mice; ^p < 0.05 compared with (+/−)-MDMA-treated mice, by Newmann–Keuls post-hoc test.

Discussion

MDMA is a widely consumed recreational drug which has rewarding, psychostimulant, and hallucinogenic properties (Green et al. 2003; Schenk 2009). Concerns about the widespread use of MDMA are not only because of its potential abuse, but also because of its ability to induce cardiac toxicity and hyperthermia (Green et al. 2003).

Previous studies, including ours, have shown that MDMA administration induces glial activation in the mouse striatum (O'Callaghan and Miller 1994; Granado et al. 2008a; Khairnar et al. 2010) associated with dopamine neuron degeneration (O'Callaghan and Miller 1994; Granado et al. 2008a,b). These studies raised the concern that, besides eliciting general toxic effects (Green et al. 2003), MDMA may also cause long-term neurotoxicity toward the dopaminergic neurons, like other amphetamine analogs, such as methamphetamine (Delle Donne and Sonsalla 1994; Gluck et al. 2001).

In this study, we provide the first demonstration of a different response of the astroglia and microglia to the acute administration of the two MDMA enantiomers. Thus, S(+)-MDMA, similar to (+/−)-MDMA, activated GFAP and CD11b in the mouse striatum, but not in the other brain areas investigated.

Interestingly, the activation of glial cells by S(+)-MDMA was associated with a hyperthermic response similar, but not identical, to that caused by (+/−)-MDMA, since elevation in body temperature by S(+)-MDMA did not reach the level of (+/−)-MDMA on administrations II and III. In contrast, R(−)-MDMA produced neither hyperthermia nor glial activation.

S(+) and R(−) enantiomers were not able to affect glial activation in brain areas other than the striatum when administered either alone or in combination, whereas racemic MDMA was able to increase microglia, but not astroglia, activation in all areas analyzed. The lack of astroglial activation in the nucleus accumbens is similar to that observed in a previous study by Granado et al. 2008ab, whereas, in our study, some microglial activation was observed in that area after (+/−)-MDMA administration. The activation of microglia observed in our study was, however, of low intensity compared with the striatum, and was observed at a different time point than the study by Granado et al. 2008ab. This finding is not completely unexpected, in the light of previous experiments suggesting that the striatum could be more sensitive to the detrimental effects of neurotoxins, than other brain areas, such as the SNc (Carta et al. 2009). Another interesting result of this study is the positive correlation between the elevation in body temperature and the activation of astroglia and microglia, which supports previous results showing that hyperthermia is one of the factors promoting glial activation and neurotoxicity by (+/−)-MDMA (Miller and O'Callaghan 1995; Colado et al. 1998; Mechan et al. 2001; Reveron et al. 2005; Touriño et al. 2010). Partial protection against MDMA toxicity on dopaminergic neurons was, in fact, obtained following exposure of animals to cooler temperatures, although the precise relationship between MDMA-mediated neurotoxicity and body temperature requires further investigation (Miller and O'Callaghan 1995; Taffe et al. 2006). Furthermore, the present results show that the two enantiomers display different activities not only in inducing neuroinflammatory responses and hyperthermia but also in eliciting motor stimulation. In fact, S(+)-MDMA, but not R(−)-MDMA, induced a motor activation similar to that obtained after (+/−)-MDMA administration. This latter result confirms previous behavioral studies showing that induction of motor and stereotyped behaviors and antagonism of catalepsy in rodents by MDMA are because of S(+)-MDMA (Hiramatsu et al. 1989; Lebsanft et al. 2005; Young and Glennon 2008; von Ameln and von Ameln-Mayerhofer 2010).

The mechanisms at the base of the differences shown by the two enantiomers in inducing glial activation are not clear; however, an important factor may be the different biochemical and pharmacokinetic properties of S(+)- and R(−)-MDMA. MDMA has an enantioselective metabolism (Meyer et al. 2002; Fantegrossi et al. 2009; Meyer and Maurer 2009), which may act differently on the biochemical factors mediating MDMA neurotoxicity, such as increase in hydroxyl radical formation and DNA fragmentation (Green et al. 2003; Granado et al. 2008a,b). Moreover, S(+)-MDMA has a faster absorption and metabolism which may produce a greater amount of toxic metabolites (Lim et al. 1993; Capela et al. 2007; Fantegrossi et al. 2009; Meyer and Maurer 2009).

Interestingly, in this study, the combined administration of S(+)-MDMA plus R(−)-MDMA produced effects similar to (+/−)-MDMA on microglial activation, whereas no additive effects were observed on the degree of astroglial activation. In fact, GFAP activation did not increase further after S(+)- plus R(−)-MDMA compared with S(+)-MDMA, remaining significantly lower compared with (+/−)-MDMA. Importantly, S(+)-MDMA activated microglia by 54% compared with (+/−)-MDMA, whereas astroglial activation was about 80% compared with (+/−)-MDMA. This might imply that the response of the astroglia, which was almost maximal after S(+)-MDMA, might be more difficult to stimulate further by combined administration of the two enantiomers. Moreover, it is worth emphasizing that the astroglia seemed to display a specific activation in the striatum after the administration of either racemic MDMA or S(+)-MDMA, whereas the other brain regions analyzed appeared unaffected. On the other hand microglia, which were less intensely activated by S(+)-MDMA, might show an additional increase after the combination of S(+)-MDMA plus R(−)-MDMA. In addition, the differences observed in the activation of astroglia and microglia might be because of the different time course of induction of the two glial populations. It is, in fact, known that microglia have a rapid induction and decline, whereas astroglia require a longer time for activation (O'Callaghan and Miller 1994; Granado et al. 2008b; Halliday and Stevens 2011). It is therefore possible that 48 h after MDMA administration, the microglia are already in the phase of declined activation, whereas the astroglia are at their maximal peak (O'Callaghan and Miller 1994; Granado et al. 2008b; Carta et al. 2009; Halliday and Stevens 2011). However, in this connection, it has to be remarked that the accurate evaluation of the time course of astroglial and microglial activation, led us to conclude that 48 h was a time point that could allow us to study these two parameters in the same animals, and possibly make direct comparisons.

These results underline the importance of chirality in the properties of MDMA and suggest that more attention should be paid to the effects produced by S(+)- and R(−)-MDMA to understand the mechanisms underlying the MDMA neuroinflammatory and neurotoxic effects. Moreover, the variety of MDMA enantiomer effects is increased by the knowledge that MDMA responses may vary, depending on the species (rodent or primate). With this regard, it is worth recalling that strong concern and ethical considerations exist on the use of MDMA in clinical experimentation, because of the toxic and schizophrenia-like effects the drug may exert (Green et al. 2003). Thus, it is still not known which is the animal species that best reproduces the effects of MDMA observed in humans. Therefore, it is extremely important to gather information on the effects of MDMA from studies performed in different animal species. In this connections, several lines of experimental evidence strongly indicate that results obtained in mice are extremely relevant to human pharmacology of drugs of abuse, including the toxic effects these drugs may elicit at the level of the brain. Notably, the mouse is one of the species mostly utilized in pre-clinical research on MDMA, also in consideration of the fact that it makes possible the use of genetically modified animals (e.g., K.O. animals) for the study of the mechanisms of toxicity mediated by the drug. All these things considered, the results of this study are highly relevant to the elucidation of the neuroinflammatory effects of MDMA, and may represent a starting point for a thorough pre-clinical investigation of MDMA-mediated brain toxicity, with potential implications for human pharmacology.

Recent studies examining neuronal degeneration and glial activation have established that astroglial and microglial activation, rather than a response to damage, are an active part of the cascade of events leading to neuronal toxicity as they play an important role in the initiation of early tissue response and in the progression of degenerative diseases (O'Callaghan and Sriram 2005; Zhang et al. 2006; Granado et al. 2008b; Halliday and Stevens 2011). In particular, in a disease such as Parkinson's disease, α-synuclein accumulation in astrocytes causes recruitment of phagocytic microglia that attack selected neurons causing neuronal degeneration (Halliday and Stevens 2011). Therefore, the present results, by showing that S(+)-MDMA is the enantiomer mostly involved in the astroglial and microglial activation induced by (+/−)-MDMA, confirm that various and complex effects of MDMA enantiomers are present, and underline the importance of studying these effects to clarify the MDMA neurotoxic potential.

Acknowledgements

The authors thank Mr. Renato Mascia, Department of Life and Environmental Sciences, University of Cagliari, for his help with the drug synthesis, Fondazione Banco di Sardegna for supporting Dr. Lucia Frau (Rep. 13/19844 del 31-01-2011), and Regione Autonoma della Sardegna for supporting Dr. Nicola Simola. (P.O.R. FSE 2007–2013).

Conflict of interest

The authors have no conflict of interest to declare.

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